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Immune system stimulating or immuno-stimulatory motifs are present in DNA and RNA

Synthetic and natural RNA molecules can contain sequence motifs that stimulate the immune system. In addition to mediating RNA interference (RNAi), silencing RNAs (siRNAs) can induce the innate immune system as well.

Pattern recognition receptors (PRRs) recognize and respond to RNA sequence motifs uniquely. In general, when designing regular siRNAs, the presence of immune-stimulating sequence motifs is not desired. Therefore, three sequence motifs, "UGUGU," "GUCCUUCAA," and "AUCGAU(N)nGGGG," need to be included in an immune-motif avoidance list. Also, U-rich sequences or sequences with biased nucleotide content, such as (G + U) >> (C + A) or with A + U or G + U rich motifs, also need to be avoided.


However, some clinical applications of RNA interference (RNAi) may benefit from siRNAs' activation of the immune system. In particular, the design of small interfering RNAs (siRNAs) with antitumor and antiviral activities.


Blood immune cells express a receptor set that helps detect pathogens and help mount an appropriate immune response. For example, Toll-like receptors (TLRs) detect DNA, RNA, or bacterial and fungal components together with modified endogenous molecules such as oxidized low-density lipoprotein (LDL) or fibrillar amyloid-β peptides. siRNAs can recruit immune receptors specialized in RNA detection, for example, TLR3 and TLR7. Double-stranded RNA can induce immune activity associated with producing a range of cytokines. Usually, this is perceived as an unwanted nonspecific effect of in vivo siRNA when administered. The use of chemically modified nucleic acids in siRNA can prevent this response, for example, using 2’-O-methyl ribonucleotides or bridged nucleic acids (BNAs).


Design of immuno-stimulatory siRNAs

Incorporating specific mismatches in the siRNA duplex's passenger strand allows the creation of an immune-stimulatory motif. The introduction of such a mismatch between bases 9 and 12 from the 5’-end of the passenger strand increases RNAi potency. However, the introduction of such a mismatch strongly depends on the siRNA sequence selected.

According to Gantier:

[1]  A uridine bulge replacing bases 9 to 12 in the passenger strand of the siRNA activates TLR8.


[2]  The fusion of a CpG DNA moiety containing phosphorothioate bonds to the 5’-end of the guide strand also increases the immune response.


[3]  Adding a triphosphate group to both strands' 5'- ends activates RIG-I.


Triphosphate moieties can be added chemically or via in-vitro transcription.


In 2015, Xu et al. identified a natural viral RNA motif in Sendai virus RNA that optimizes sensing of viral RNA by RIG-I. This RNA motif named DVG70-114 is essential for the potent immunostimulatory activity of 5′-triphosphate-containing Send virus (SeV) iDVGs. According to Xu et al., DVG70-114 enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs. The motif retains its stimulatory potential when transferred to otherwise inert viral RNA. In vitro analysis showed that DVG70-114 augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization. The study defined a new natural viral PAMP enhancer motif that promotes viral recognition by Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and confers potent immunostimulatory activity to viral RNA.

Table 1: RNA sequence motifs that stimulate immune responses

Sequence

Signaling pathway

Cytokines

 

 

 

UGUGU

Immune stimulating. Toll-like receptor (TLR) 8

Interferon (IFN)-a

GUCCUUCAA

Immune stimulating. TLR 7 and 8.

IFN-a

AUCGAU(N)nGGGG

Immune stimulating.

 

U-rich sequences. Uracil repeats

Immune stimulating. TLR 7.

IFN-a. interleukin-6, TNF-a

(G+U)>>(C+A)

Immune stimulating.

 

A+U or G+U rich

Immune stimulating.  7 and 8.

IFN-a. tumor necrosis factor (TNF)-a

UGGC

Reduced cell viability.

 

GGGG; UUUU, CCCC, AAAA

Low sequence complexity.

 

AU-rich

Activate TLR8.

 

GU-rich

Activate TLR7 and TLR8.

 

A- and U- rich

Stimulate TLR8. Triggers INFNa and TNFa production.

 

UUGU, GUUC, GUUU, UUUC, UGUU, or UCUC 

Activating human TLR7/8 by inducing IFN-α and proinflammatory cytokines and chemokines from cells expressing only TLR7 or both TLR7 and TLR8.

 

AUGU, UAUA, AUAU, AUAC, UAUU, UUAU, CUAC, GUAC, or UAUC

Induce the strongest TNF-α production by lacking substantial IFN-α secretion and revealed target cell and receptor selectivity by stimulating monocytes and mDCs, but not pDCs.

 

GU-rich ORNs 

Induce substantially stronger IFN-α production from human PBMCs than a poly(U) ORN of the same length. 

 

Blunt ended

Retinoic acid-inducible gene- I (RIG-I).

Type I IFN, p56

5’-Triphosphate

RIG-I.

INF-α, INF-β

MicroRNA-like small interfering RNA

TLR 7 and 8.

IFN-α, TNF-α

Unmethylated CpG motifs

Immune stimulation.

Activate natural killer cells, induced interferon γ (IFNγ).


The cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) also activate human B cells and dendritic cells (DCs).  These properties suggest a potential use as an adjuvant to enhance vaccine efficacy. 


Reference

Lopez C.; Method and composition of stimulating immune response using potent immunostimulatory RNA motifs: PubChem [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004. PubChem Patent Summary for US-10624964-B2. [US Patent]

Gantier, M.P.; Strategies for designing and validating immunostimulatory siRNAs. In Debra J. Taxman (ed.); siRNA design: Method and Protocols. MMB 942. Chapter 10 pp 179. Humana Press 2013.


Forsbach A, Nemorin JG, Montino C, Müller C, Samulowitz U, Vicari AP, Jurk M, Mutwiri GK, Krieg AM, Lipford GB, Vollmer J. Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses. J Immunol. 2008 Mar 15;180(6):3729-38. doi: 10.4049/jimmunol.180.6.3729. PMID: 18322178. [
Jimmunol]

Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol. 2002;20:709-60. [Annual Reviews]

Zhongji Meng and Mengji Lu; RNA Interference-Induced Innate Immunity, Off-Target Effect, or Immune Adjuvant? Front. Immunol., 23 March 2017 [Frontiersin]

Netea, M.G., Domínguez-Andrés, J., Barreiro, L.B. et al. Defining trained immunity and its role in health and disease. Nat Rev Immunol 20, 375–388 (2020). [
nature]

Xu J, Mercado-López X, Grier JT, Kim WK, Chun LF, Irvine EB, Del Toro Duany Y, Kell A, Hur S, Gale M Jr, Raj A, López CB. Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I. mBio. 2015 Oct 6;6(5):e01265-15.  [PMC]

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